Non-pyrogenic, endotoxin-frei stroma-free tetrameric hemoglobin

ABSTRACT

A non-pyrogenic, endotoxin-free, stroma-free, blood substitute capable of being used in humans or other mammals to provide oxygen to tissue and to deliver carbon dioxide to the lung and a process for its preparation are described.

FIELD OF THE INVENTION

[0001] The invention relates to a blood substitute capable of being usedin humans or other mammals to provide oxygen to tissue and to delivercarbon dioxide to the lung. More specifically, the invention provides aprocess for producing a cross-linked tetrameric hemoglobin preparationthat is non-pyrogenic, endotoxin-free and stroma-free such that it iscapable of in vivo use in a human or other mammals.

BACKGROUND OF THE INVENTION

[0002] It is not always practical or safe to transfuse a patient withdonated blood. One of the limitations on the use of blood in anemergency setting is the requirement to type and cross-match the bloodto minimize the risk of transfusion rejection. Saline cross-matchingrequires at least 10 minutes and a complete type and cross-match cantake up to an hour. Furthermore, the risk of HIV transmission has beenestimated to be 1 in 500,000 units of blood and the risk of hepatitis Ctransmission has been estimated to be 1 in 3,000 units. Schreiber etal., N. Engl. J. Med. 334:1685-90 (1996), incorporated by reference inits entirety.

[0003] Therefore, a red blood cell (“RBC”) substitute has long beensought after. To be effective as a substitute for red blood cells, anRBC substitute ideally will meet several requirements. It must bevirus-free, non-toxic and non-immunogenic and it should havesatisfactory oxygen carrying capacity and circulatory persistence topermit effective oxygenation of tissues. Preferably, the oxygen affinityshould be close to that of whole blood (p50=28 mmHg at 37° C.) (Ogden,J. E. et al., Vox Sang 69:302-308 (1995)).

[0004] Three general classes of blood substitutes have beeninvestigated: perfluorcarbons, liposome encapsulated hemoglobin andhemoglobin derivatives. Perfluorcarbons are inert chemically synthesizedcompounds that dissolve oxygen. Perfluorcarbons suffer from thedisadvantage that they are immiscible in aqueous solutions and thus mustbe emulsified with lipids before being introduced into the blood stream.Liposomes suffer from structural rigidity, and from a low effectivehemoglobin concentration.

[0005] Because hemoglobin mediates the delivery of oxygen from the lungsto the tissues, purified hemoglobin has been extensively investigated asa possible blood substitute. Hemoglobin is reported to be approximately97% pure inside the red blood cell. Human hemoglobin is a protein havinga molecular weight of 64 kD, and it consists of four subunits, two alphapolypeptide chains and two beta polypeptide chains. Each of the subunitscontains a single iron-containing heme prosthetic group that binds andreleases oxygen. Hemoglobin exhibits cooperative binding of oxygen bythe four subunits of the hemoglobin molecule, and this cooperativelyfacilitates oxygen transport. When hemoglobin binds oxygen, it shiftsfrom the high energy “tense” or “T” state (deoxygenated) to the lowerenergy “relaxed” or “R” state (oxygenated). Human alpha and beta globingenes have been cloned and sequenced (Liebhaber et al., Proc. Natl.Acad. Sci. (U.S.A). 77:7054-58 (1980); Marotta et al., J. Biol. Chem.252:5040-43 (1977); and Lawn et al., Cell 21:647 (1980), all of whichare incorporated by reference in their entirety).

[0006] Because of its natural role in oxygen delivery, hemoglobin haslong been the target of efforts to develop a blood substitute. Themembranes of red blood cells, which are referred to as ghosts or stroma,contain all of the blood type antigens. Rabiner et al. firstdemonstrated that some of the toxic properties of hemolyzed red bloodcells were related to the membrane (stroma) of red blood cells and theirrelated lipids. Rabiner et al., J. Exp. Med. 126:1127 (1967). Themembranes are destroyed by freezing so that storage requirements forblood require climate controlled refrigeration. In addition, many of thehuman viral diseases transmitted through blood transfusions adhere tothe stroma of red blood cells. Thus, in view of the immunogenicproperties of the cell membranes of red blood cells, and the problems ofviral contamination, stroma-free hemoglobin (“SFH”) was initiallyselected for therapeutic research.

[0007] An effective stroma-free hemoglobin blood substitute therapywould offer several advantages over conventional blood replacementtherapies. Significantly, the use of stroma-free hemoglobin bloodsubstitutes is predicted to reduce the extent and severity of undesiredimmune responses, and the risk of transmission of viral diseases,including hepatitis and HIV. Moreover, in contrast to the limitedstorage capacity of erythrocytes, stroma-free hemoglobin bloodsubstitutes are predicted to exhibit an extended shelf life, and torequire less rigorous storage facilities.

[0008] However, several problems plagued stromal-free hemoglobinisolation procedures. In particular, it was found that the SFH must befree of any part of the red cell membrane as it is the red cell membranewhich causes the immune response. Thus complete purification from thestroma was required.

[0009] Additionally, once outside of the red blood cell, hemoglobin wasfound to have such a high affinity for oxygen that it would not releaseit to the tissues under physiological conditions. SFH was also found topossess only a limited half-life in the body, and to be rapidly clearedfrom the blood by glomular filtration. This disrupts the ability of thekidney to concentrate urine and results in the rapid removal ofhemoglobin from the intravascular volume. Excessive filtration of thealpha/beta subunit by the glomerulus in the kidney can cause osmoticdiuresis. In vivo, the retention time of stroma-free human hemoglobin ison the order of 14 hours. De Venuto et al., Transfusion 17:555 (1977).

[0010] The rapid clearing of SFH by the kidney is a consequence of itsquaternary molecular arrangement. As indicated above, natural hemoglobinis composed of a tetrameric arrangement of alpha and beta polypeptidechains. Within the RBC, the association of the alpha chain with itscorresponding beta chain is very strong and does not disassociate underphysiological conditions. The association of one alpha/beta dimer withanother alpha/beta dimer, however, is fairly weak and outside of theRBC, the two dimers disassociate even under physiological conditions.Upon disassociation, the dimer is filtered through the glomerulus.

[0011] To avoid such removal, SFH has been chemically cross-linked toform a stable tetramer. Several chemical agents have been used tocross-link hemoglobin alpha/beta dimers and prevent their filtration bythe glomerulus into the urine, and yet maintain the oxygen transportproperties of native hemoglobin. Bis dibromo salicyl fumarate (BDBF) isan activated diester of fumaric acid that has been used as across-linker to cross-link hemoglobin (Tye, U.S. Pat. No. 4,529,719,hereby incorporated by reference in its entirety). Fumaric acid is afour carbon straight chain unsaturated trans 2,3 dicarboxylic acid whichis capable of interacting with the aspirin binding site of bothalpha/beta dimers. This maintains the two dimers in proper orientationfor cross-linking with lysine residues. A slight molar excess of BDBFcross-linker to hemoglobin (1.2:1.0), under sub-optimum conditions, hasbeen reported to yield 70% cross-linked hemoglobin molecules.

[0012] The tetrameric structure of hemoglobin provides a binding sitefor 2,3-diphosphoglycerate. Inside red blood cells, the binding of2,3-diphosphoglycerate to hemoglobin decreases the hemoglobin's oxygenaffinity to a level compatible with oxygen transport. The binding of2,3-diphosphoglycerate to hemoglobin is very weak and requires very highconcentrations (i.e., concentrations approaching 1M or more) in order tomodify the affinity of hemoglobin for oxygen. Thus, when the red bloodcells are ruptured to produce SFH, the 2,3-diphosphoglycerate is notretained in close proximity to the hemoglobin and disassociates from thehemoglobin. As a consequence, unless further modified, SFH exhibits ahigher affinity for oxygen than does hemoglobin in RBCs. The increasedaffinity of the SFH for oxygen is quite significant since, underphysiological conditions, it is unable to release the bound oxygen tothe tissues. Bovine hemoglobin does not require 2,3-DPG to maintain ap50 for oxygen in the range of 30 mm.

[0013] Cross-linking the alpha or beta chains of hemoglobin will preventdisassociation of the tetramer. It is the disassociation of R statehemoglobin into dimers which allows hemoglobin in the plasma to befiltered by the glomerulus into urine and removed by haptoglobin intothe reticuloendothelial system.

[0014] The tetrameric structure of T state deoxyhemoglobin has increasedstability from six ionic bonds and while in the T state, hemoglobin iseffectively prevented from disassociating into dimers. In thisconformation, the beta cleft contact area between the two beta chains(also known as the beta pocket, phosphate pocket, and2,3-diphosphoglycerate binding site) in deoxyhemoglobin is substantiallydifferent than in oxyhemoglobin. The changed conformation of the betacleft in the T state is believed to explain the decreased oxygenaffinity stabilized by 2,3-diphophoglycerate. The T state of hemoglobinis stable and resistant to denaturation. Thus, cross-linking the SFHaddresses both the problem of oxygen affinity and the problem of rapidfiltration by the kidney.

[0015] Other blood substitutes have been described (Tye, U.S. Pat. No.4,529,719), and may be employed in cases of acute and severe blood loss.However, a need still exists for a blood substitute that exhibits evenlower pyrogenicity, and which may therefore be employed in non-acutecases or in cases of less severe need, or for chronic, long term ornon-emergency transfusion use. The present invention provides such animproved blood substitute.

SUMMARY OF THE INVENTION

[0016] This invention is directed to a method for producing a purifiedpreparation of an endotoxin-free, stroma-free, cross-linked tetramerichemoglobin, and to an endotoxin-free, stroma-free, cross-linkedtetrameric hemoglobin.

[0017] In detail, the invention provides, a non-pyrogenic,endotoxin-free, oxygen-free, stroma-free, cross-linked tetramerichemoglobin. The invention particularly concerns the embodiments in whichthe hemoglobin has been cross-linked with bis dibromo salicyl fumarateand/or has been modified by reaction with pyridoxal-5′-phosphate. Theinvention particularly concerns the embodiments in which the hemoglobinis human hemoglobin, or is bovine or porcine hemoglobin. Such moleculesmay be obtained from any of a variety of sources (for example, fromanimal sources, via recombinant technology, from chemical synthesis,etc.).

[0018] The invention also provides a non-pyrogenic, endotoxin-free,oxygen-free, stroma-free, cross-linked tetrameric hemoglobin.

[0019] The invention further provides a blood substitute compositioncomprising a preparation of non-pyrogenic, endotoxin-free, stroma-free,cross-linked tetrameric hemoglobin, and a pharmaceutically acceptablecarrier.

[0020] The invention further provides a method of supplementing theblood of a mammal which comprises administering to the mammal a bloodsubstitute composition comprising a preparation of non-pyrogenic,endotoxin-free, stroma-free, cross-linked tetrameric hemoglobin and apharmaceutically acceptable carrier.

[0021] The invention further provides a preparation of non-pyrogenic,endotoxin-free, stroma-free, cross-linked tetrameric hemoglobin producedby the process comprising the steps of:

[0022] (A) removing endotoxin from a preparation containing red bloodcells;

[0023] (B) removing oxygen from the preparation containing red bloodcells; and

[0024] (C) lysing red blood cells;

[0025] or the steps of

[0026] (A′) removing endotoxin from a preparation containing red bloodcells;

[0027] (B′) lysing red blood cells; and

[0028] (C′) removing oxygen from hemoglobin of the lysed red bloodcells.

[0029] The invention particularly concerns the sub-embodiments whereinin process step (B) or (C′), the oxygen is removed by centrifuging thered blood cells under vacuum.

[0030] The invention additionally concerns the sub-embodiment whereinthe process for preparing such non-pyrogenic, endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin additionally comprisesthe steps of:

[0031] (D) separating hemoglobin from the stroma of the lysed red bloodcells; and

[0032] (E) cross-linking the separated hemoglobin.

[0033] The invention particularly concerns the sub-embodiment whereinprocess step (A) additionally comprises washing surfaces and equipmentthat will come into contact with the cross-linked hemoglobin with adilute solution of hemoglobin.

[0034] The invention particularly concerns the sub-embodiments whereinprocess step (B) or (C′) comprises subjecting the red blood cellpreparation to a vacuum sufficient to remove oxygen from thepreparation. The invention further concerns the sub-embodiment whereinprocess step (B) additionally comprises centrifuging a solution of thecells under vacuum at a speed sufficient to produce a force greater thanthe surface tension of the solution.

[0035] The invention particularly concerns the embodiment wherein thepreparation of endotoxin-free, stroma-free, cross-linked tetramerichemoglobin additionally contains a pharmaceutically acceptable carrier.

[0036] The invention also provides a method for producing anon-pyrogenic, endotoxin-free, stroma-free, cross-linked tetramerichemoglobin comprising the steps of:

[0037] (A) removing endotoxin from a preparation containing red bloodcells;

[0038] (B) removing oxygen from the preparation containing red bloodcells; and

[0039] (C) lysing red blood cells;

[0040] or the steps of

[0041] (A′) removing endotoxin from a preparation containing red bloodcells;

[0042] (B′) lysing red blood cells; and

[0043] (C′) removing oxygen from hemoglobin of the lysed red bloodcells.

[0044] The invention also concerns the embodiments of such methodswherein in step (B) or (C′), the oxygen is removed by centrifuging thered blood cells under vacuum.

[0045] The invention also concerns the embodiment of such method whereinthe method additionally comprises the steps of:

[0046] (D) separating hemoglobin from the stroma of the lysed red bloodcells; and

[0047] (E) cross-linking the separated hemoglobin.

[0048] The invention particularly concerns the sub-embodiment whereinmethod step (A) additionally comprises washing surfaces and equipmentthat will come into contact with the cross-linked hemoglobin with adilute solution of hemoglobin.

[0049] The invention particularly concerns the sub-embodiments whereinmethod step (B) or (C′) comprises subjecting the red blood cellpreparation to a vacuum sufficient to remove oxygen from thepreparation. The invention further concerns the sub-embodiments whereinmethod step (B) or (C′) additionally comprises centrifuging a solutionof the cells under vacuum at a speed sufficient to produce a forcegreater than the surface tension of the solution.

[0050] The invention also provides a method of increasing the oxygencarrying capacity of an individual which comprises administering to theindividual a non-pyrogenic, endotoxin-free, stroma-free, cross-linkedtetrameric hemoglobin. The invention particularly concerns theembodiment wherein the non-pyrogenic, endotoxin-free, stroma-free,cross-linked tetrameric hemoglobin is administered by transfusion orinjection.

[0051] The invention further concerns the method for increasing anindividual's oxygen carrying capacity, wherein the non-pyrogenic,endotoxin-free, stroma-free, cross-linked tetrameric hemoglobin isproduced by a process comprising the steps:

[0052] (A) removing endotoxin from a preparation containing red bloodcells;

[0053] (B) removing oxygen from the preparation containing red bloodcells; and

[0054] (C) lysing red blood cells;

[0055] or the steps of

[0056] (A′) removing endotoxin from a preparation containing red bloodcells;

[0057] (B′) lysing red blood cells; and

[0058] (C′) removing oxygen from hemoglobin of the lysed red bloodcells.

[0059] The invention particularly concerns the sub-embodiments whereinin process step (B) or (C′), the oxygen is removed by centrifuging thered blood cells under vacuum.

[0060] The invention additionally concerns the sub-embodiment whereinthe process for preparing such non-pyrogenic, endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin additionally comprisesthe steps of:

[0061] (D) separating hemoglobin from the stroma of the lysed red bloodcells; and

[0062] (E) cross-linking the separated hemoglobin.

[0063] The invention particularly concerns the sub-embodiment whereinprocess step (A) comprises washing surfaces and equipment that will comeinto contact with the cross-linked hemoglobin with a dilute solution ofhemoglobin.

[0064] The invention particularly concerns the sub-embodiments whereinprocess step (B) or (C′) comprises subjecting the red blood cellpreparation to a vacuum sufficient to remove oxygen from thepreparation. The invention further concerns the sub-embodiments whereinprocess step (B) or (C′) additionally comprises centrifuging a solutionof the cells under vacuum at a speed sufficient to produce a forcegreater than the surface tension of the solution.

[0065] The invention also provides a container containing anon-pyrogenic, endotoxin-free, stroma-free, cross-linked tetramerichemoglobin composition. The invention particularly concerns theembodiments in which the container is an anoxic container composed ofpolyethylene terephthalate, or is an implantable delivery device thatdelivers a non-pyrogenic, endotoxin-free, stroma-free, cross-linkedtetrameric hemoglobin composition to a recipient.

[0066] The invention particularly contemplates that the non-pyrogenic,endotoxin-free, stroma-free, cross-linked tetrameric hemoglobincontained in the container is produced through the process comprisingthe steps of:

[0067] (A) removing endotoxin from a preparation containing red bloodcells;

[0068] (B) removing oxygen from the preparation containing red bloodcells;

[0069] (C) lysing red blood cells;

[0070] (D) separating hemoglobin from the stroma of the lysed red bloodcells; and

[0071] (E) cross-linking the separated hemoglobin;

[0072] or

[0073] (A′) removing endotoxin from a preparation containing red bloodcells;

[0074] (B′) lysing red blood cells;

[0075] (C′) removing oxygen from hemoglobin of the lysed red bloodcells;

[0076] (D′) separating hemoglobin from the stroma of the lysed red bloodcells; and

[0077] (E′) cross-linking the separated hemoglobin.

[0078] The invention particularly concerns the embodiments wherein thehemoglobin of such non-pyrogenic, endotoxin-free, oxygen-free,stroma-free, cross-linked tetrameric hemoglobin has been cross-linkedwith bis dibromo salicyl fumarate, and/or wherein the hemoglobin hasbeen modified by reaction with pyridoxal-5′-phosphate.

[0079] The invention particularly concerns the embodiments wherein thehemoglobin of such non-pyrogenic, endotoxin-free, oxygen-free,stroma-free, cross-linked tetrameric hemoglobin is human, bovine orporcine hemoglobin. Such molecules may be obtained from any of a varietyof sources (for example, from animal sources, via recombinanttechnology, from chemical synthesis, etc.).

DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention concerns a non-pyrogenic, endotoxin-freepurified preparation of cross-linked, stroma-free hemoglobin. As usedherein, a preparation of hemoglobin is said to be “non-pyrogenic” if itmay be administered into an individual of the same species as that fromwhich the hemoglobin was derived (i.e., a human for human-derivedhemoglobin, etc.) without causing an immunologic or pyrogenic reaction(such as inflammation, agglutination, clotting, etc.). Any of a varietyof assays may be employed to demonstrate the non-pyrogenicity of thecompositions of the present invention: interleukin-6 and other cytokineinduction (Pool, E. J. et al., J. Immunoassay 19:95-111 (1998), Poole,S. et al., Dev. Biol. Stand. 69:121-123 (1988)); human monocytoid cellline assays (Eperon, S. et al., J. Immunol. Meth. 207:135-145 (1997),Taktak, Y. S. et al., J. Pharm. Pharmacol. 43:578-582 (1991)); theLimulus Amoebocyte Lysate (LAL) test (Fujiwara, H. et al., YakugakuZasshi 110:332-40 (1990), Martel F. et al., Rev Fr TransfusImmunohematol 28:237-250 (1985)) and the rabbit pyrogen test (Bleeker W.K. et al., Prog Clin Biol Res 189:293-303 (1985), Simon, S. et al., Dev.Biol. Stand. 34:75-84 (1977); Allison, E. S. et al., Clin. Sci. Mol.Med. 45:449-458 (1973)); all herein incorporated by reference. Therabbit pyrogen test is the preferred pyrogenicity assay.

[0081] As used herein, a preparation of hemoglobin is said to be“stroma-free” if the hemoglobin has been treated to remove substantiallyall stromal material, such that the preparation no longer exhibits theimmunoreactivity to blood type antigens characteristic of RBCs.Stroma-free hemoglobin thus substantially lacks the toxic and/orpyrogenic properties associated with preparations of hemolyzed red bloodcells, and thus can be administered to an individual without causingtoxicity or inflammatory reaction. As used herein, a preparation ofhemoglobin is said to be “endotoxin-free” if the hemoglobin has beentreated to remove substantially all endotoxin. Thus, for the purposes ofthe present invention, endotoxin-free hemoglobin has an amount ofendotoxin ranging from 0-10%, and more preferably from 0-1%, of theamount of endotoxin present in USP water. In a preferred method forforming such endotoxin-free, stroma-free cross-linked hemoglobin, thehemoglobin is deoxygenated to render it “oxygen-free.” As used herein, apreparation of hemoglobin is said to be “oxygen-free” if the hemoglobinhas been treated to remove substantially all oxygen. Oxygen-freehemoglobin thus is substantially or completely in the higher energy“tense” or “T” configuration.

[0082] Although DBDF cross-linked hemoglobin appeared to be verypromising based upon it's initial characterization, prior preparationsof DBDF cross-linked hemoglobin were found to lack clinical efficacy.The present invention has identified several of properties DBDFcross-linked hemoglobin that have reduced the clinical efficacy of themolecule, and the present invention provides an improved DBDFcross-linked hemoglobin (FXSFH) for overcoming the deficiencies of theprior preparations. Principally, the hemoglobin derivative must beprepared in the absence of oxygen. Inorganic phosphate, which bindstightly to the hemoglobin molecule and interferes with the cross-linkingreaction, must be removed to increase yield. Endotoxins, which bindtightly to the hemoglobin molecule and become a hepatic toxin when thehemoglobin is metabolized, must not be allowed to contact thehemoglobin.

[0083] Hemoglobin that has been purified from the stroma in the absenceof oxygen is referred to as “stroma-free deoxyhemoglobin” (“dSFH”) andis in the higher energy “tense” or “T” configuration. Hemoglobincross-linked in the presence of oxygen binds oxygen very tightly andwill not release the oxygen to the tissue under physiologicalconditions. Removal of oxygen from the hemoglobin solution prior toreaction with DBDF has been very difficult. Concentrated proteinsolutions froth and foam when oxygen is removed by bubbling nitrogenthrough the solution or removal of oxygen by placing a vacuum over thesolution. One of the problems the art has faced is the lack of anendpoint to discern when all of the oxygen has been removed. When oxygenis removed, there is a change in the pH of the solution which must becompensated for while the reaction occurs. Foaming can be controlledbriefly by the addition of a surfactant, but that can introduceadditional endotoxins into the final product. Foaming causes denaturedprotein; surfactant adds endotoxin.

[0084] I. Preferred Method for Producing FXSFH

[0085] A. Sources of Hemoglobin

[0086] The cross-linked stroma-free hemoglobin (“FXSFH”) of the presentinvention can be prepared in a single working day with a properlyequipped laboratory. Although the chemical reaction itself is extremelyrapid, much of the time is spent concentrating hemoglobin, removingreaction products, or equilibrating with physiological buffers.

[0087] Stroma-free hemoglobin may be obtained from a variety ofmammalian sources, such as, for example, human, bovine, ovine, orporcine sources. Alternatively, the stroma-free hemoglobin of thepresent invention may be synthetically produced by a bacterial, or morepreferably, by a yeast, mammalian cell, or insect cell expression vectorsystem (Hoffman, S. J. et al., U.S. Pat. No. 5,028,588 and Hoffman, etal., WO 90/13645, both herein incorporated by reference. Alternatively,hemoglobin can be obtained from transgenic animals; such animals can beengineered to express non-endogenous hemoglobin (Logan, J. S. et al. PCTApplication No. PCT/US92/05000; Townes, T. M. et al., PCT ApplicationNo. PCT/US/09624, both herein incorporated by reference).

[0088] Preferably, the stroma-free hemoglobin of the present inventionis isolated from bovine or human source, and most preferably a humansource.

[0089] Such hemoglobin, whether derived from an animal, synthetic orrecombinant, may be composed of the “naturally existing” hemoglobinprotein, or may contain some or be entirely composed of, a mutanthemoglobin protein. Preferred mutant hemoglobin proteins include thosewhose mutations result in more desirable oxygen binding/releasecharacteristics. Examples of such mutant hemoglobin proteins includethose provided by Hoffman, S. L. et al. (U.S. Pat. Nos. 5,028,588 and5,776,890) and Anderson, D. C. et al. (U.S. Pat. Nos. 5,844,090 and5,599,907), all herein incorporated by reference.

[0090] B. Cleansing of Membranes and Equipment

[0091] Since endotoxins are highly undesirable, it is preferred that allmembranes, and equipment used to produce the FXSFH of the presentinvention be cleansed in a manner sufficient to cause the removal orelimination of endotoxin that may be present on such materials andequipment.

[0092] Preferably, such cleansing is accomplished by pre-washingsurfaces and equipment that will come into contact with the FXSFH of thepresent invention using a dilute solution of hemoglobin. Such a solutionserves to bind endotoxin and hence to remove residual endotoxin that maybe present on such membranes or equipment. The dilute solution ofhemoglobin is preferably discarded after each use.

[0093] C. Removal of Oxygen

[0094] The erythrocyte preparation that is to be used as the source ofthe hemoglobin of the present invention is treated under conditionssufficient to remove oxygen present in the preparation. One aspect ofthe present invention concerns an improved process for removing oxygenfrom SFH preparations. Such deoxygenation may be performed either priorto, or subsequent to erythrocyte membrane disruption.

[0095] The removal of contaminating oxygen during the hemoglobinisolation is probably the most critical step in the formation of FXSFH.This step is difficult to accomplish, and most investigators erroneouslybelieve that merely by bubbling nitrogen through the solution for 15-30minutes they will have removed substantially all of the oxygen present.Additionally, investigators do not measure the levels of oxygen in thesolution nor do they estimate the amount of “T” state hemoglobin presentin their reaction vessels.

[0096] The extent of deoxygenation can be measured by gas chromatograph,zirconium-based detector (e.g., a “MOCON” analyzer (Mocon, Minneapolis,Minn.), by measuring pO₂ or by measuring the spectral shift that ischaracteristic of deoxyhemoglobin formation.

[0097] 1. Removal of Oxygen Prior to Erythrocyte Membrane Disruption

[0098] A preferred method is to remove the oxygen from the red bloodcells that have been washed in isotonic saline prior to hypotonic lysis.The cells still have a large intracellular concentration of 2,3-DPG andthus a lower affinity for oxygen. The cell membrane prevents thehemoglobin protein from foam denaturation.

[0099] In this embodiment, oxygen removal is effected by subjecting theerythrocyte preparation to a vacuum sufficient to remove oxygen from thepreparation. In a highly preferred embodiment, oxygen removal isaccomplished by agitating, or even more preferably, by centrifuging, thecells while under vacuum. Such treatment takes advantage of the factthat oxygen has a looser affinity for hemoglobin contained withincellular membranes than it does for free hemoglobin. By conducting theoxygen removal prior to erythrocyte membrane disruption (i.e., while thehemoglobin is within intact erythrocytes) undesired side effects, suchas bubbling or foaming of the protein, and/or its denaturation areavoided or minimized. Centrifugation should be sufficiently extensive toallow deoxygenation, but sufficiently gentle to avoid unacceptable lysisof fragile erythrocytes. Heat may be provided to prevent the solutionfrom freezing. In general, it is preferred to keep the cells at roomtemperature and to employ a vacuum sufficient to equal the vaporpressure of water at the solution of the temperature. After the removalof oxygen, all further steps are conducted in the absence of oxygen. Ina preferred embodiment, such further steps are conducted under nitrogen(or other inert gas) positive pressure in the absence of oxygen.

[0100] Emphasis is to be made that while this embodiment provides asignificantly improved method for deoxygenating hemoglobin, care must bemade to be very thorough in the removal of all traces of oxygen.

[0101] Cells that have been treated in the above manner are then lysedby addition of approximately 10 volumes of deoxygenated, endotoxin-freewater. The water may be deoxygenated by application of a vacuum andwarming of the solution, preferably to its boiling point. The red celllysis is allowed to proceed and the stroma is subsequently removed byultrafiltration. After such treatment, the temperatures are equilibratedbelow room temperature.

[0102] All subsequent steps are carried out in the absence of oxygen,maintained by what ever means is desired. A preferred method is the useof a nitrogen positive pressure environmental glove box. Other inertgases (e.g., argon) may be equivalently employed in lieu of nitrogen.

[0103] 2. Removal of Oxygen Subsequent to Erythrocyte MembraneDisruption

[0104] In an alternate preferred embodiment, the erythrocyte membranesare disrupted prior to the deoxygenation procedure. In this embodiment,the SFH has been separated from the stroma prior to deoxygenation, andhas also been separated from the high concentration of 2′3′ DPG foundwithin red cells. Due to the lowered (or absent) 2′3′ DPGconcentrations, this SFH will have a relatively high affinity foroxygen. As such, it is subject to foam denaturation during the removalof oxygen.

[0105] Accordingly, the erythrocyte preparation is preferably subjectedto hypotonic lysis, and the lysate or retentate is then filtered toremove the stroma. Oxygen contaminating the resulting material isremoved by vacuum, and more preferably by vacuum centrifugation. The SFHused may be an ultrafiltrate obtained from the removal of stroma(dilute) or a retentate from the ultrafiltration of the second stageultrafiltration conducted to concentrate the hemoglobin to approximately10% (w/v). Either of these solutions of SFH can be readily deoxygenatedby applying a vacuum sufficient to equal the partial pressure of waterat the temperature of the solution, while the solution is centrifuged ata speed sufficient to produce a force greater than the surface tensionof the solution. These are generally low speeds and can easily be metwith preparatory centrifuges, or those of a continuous flow variety. Itis desirable to consider the geometry of the containers of the SFH toinsure that there will be adequate surface area for gas exchange, andthat the temperature can be maintained and the solution not allowed tofreeze.

[0106] The dSFH prepared in the manner described above is preferablymaintained in its inert environment and the pH of the preparation ispreferably adjusted to a range between 6.0 and 8.5, and most preferablyabout pH 7.2. The pH of the solution is preferably adjusted using dilute0.1 N HCl or 0.1 N NaOH that has been previously determined to be freeof endotoxin.

[0107] Where dilution, suspension, or addition of water (includingbuffers, etc.) for other purposes is desired, such water should bedeoxygenated and be free of endotoxin. The water may be deoxygenated asdescribed above. All subsequent steps are carried out in the absence ofoxygen, maintained by what ever means is desired. As indicated above, apreferred method involves the use of a nitrogen positive pressureenvironmental glove box, however, other inert gases may be equivalentlyemployed.

[0108] D. Membrane Disruption

[0109] Hemoglobin may be released from the erythrocyte by hypotoniclysis in twenty volumes of deionized water. Other methods of erythrocytelysis, such as “slow hypotonic lysis” or “freeze thaw”, may also beemployed. See, e.g., Chan et al., J. Cell Physiol. 85:47-57 (1975),incorporated by reference in its entirety. Under one of the preferredembodiments of the present invention, the cells are lysed by flow mixingdeoxygenated red blood cells in isotonic saline with 12 volumes ofdeoxygenated, deionized, endotoxin-free water and subjecting the cellsto gentle agitation.

[0110] In order to collect the erythrocytes, the deoxygenated bloodsamples are washed several times with an isotonic solution and theplasma is separated by centrifugation at 3,000 rpm. Preferably, theisotonic solution used is a saline solution. Preferably, the cells arewashed at least three times, rinsed between each centrifugation, andresuspended in a final volume of an equal volume of isotonic solution.

[0111] The use of a sonicator is discouraged as it makes membranespheres (often referred to as “dust”). Agitation methods suitable foruse in the present invention include a magnetic stir bar and amechanical rocker or shaker.

[0112] E. Separation of Stroma From Hemoglobin

[0113] The stroma may be removed by ultrafiltration of the oxygen-freehemolysate over a 0.5μ filter which retains the cellular components andpasses the hemoglobin. Alternatively, the cellular debris is removed bysubsequent filtration through a 0.2μ filter. Ultrafiltration membranessuitable for use in the present invention are commercially availablefrom, for example, Millipore Corporation. This step is preferablyperformed at 4° C. as rapidly as possible after hemolysis of theerythrocyte, and in an oxygen-free environment. It is understood thatother methods of removing the stroma may also be used in the presentinvention.

[0114] F. Removal of Phosphate Ion

[0115] Bucci et al. (U.S. Pat. No. 5,290,919) have reported that removalof organic phosphates, e.g., 2,3-diphosphoglycerate, is necessary inhuman hemolysates because the site of the cross-linking reaction is thesame as that occupied by 2,3-diphosphoglycerate in hemoglobin.Accordingly, in a preferred embodiment, the dSFH that has passed throughthe filter is then treated to exchange phosphate for chloride. For thispurpose, the dSFH is passed, in the absence of oxygen, through an ionexchange column that has been previously prepared and equilibrated withchloride. Efficacy of this step is measured by total inorganic phosphateanalysis. Suitable ionic resins are commercially available fromPharmacia and Waters. The ionic resin removes phosphate that competesfor the aspirin binding site during the reaction with BDBF.

[0116] G. Concentration of SFH

[0117] After such treatment, the stroma-free hemolysate is concentratedby a membrane that does not allow for the passage of hemoglobin.Preferably, the stroma-free hemolysate is concentrated using a filterhaving a 30,000 MW cut-off. Preferably, the stroma-free hemolysate isconcentrated to a 1%-20% (g/l) solution. More preferably, thestroma-free hemolysate is concentrated to about 5 to about 10%. Mostpreferably, the stroma-free hemolysate is concentrated to about 10%.

[0118] The concentrated solution should be equilibrated with buffer andthe pH should be adjusted. Preferably, the pH is adjusted to a pH of7.40. However, a pH of between about 6.5 and about 8.5 can be used inthe present invention.

[0119] H. Cross-Linking with BDBF and Reaction with PLP

[0120] The completely deoxygenated, phosphate-free SFH is cross-linkedto form tetrameric hemoglobin. In a preferred embodiment, the dSFH iscross-linked with bis dibromo salicyl fumarate (BDBF) (Tye, U.S. Pat.No. 4,529,719, hereby incorporated by reference in its entirety). Toaccomplish this, BDBF cross-linker is added, with stirring to providemixing, to the dSFH preparation at a molar ratio of BDBFcross-linker:dSFH of greater than 1:1. Prior to such addition, the pH ofthe dSFH preparation is adjusted to match that of the BDBF The pH of thereaction mixture is carefully maintained by the addition of acid or basesince the solution is not buffered. This reaction is very quick, taking5 minutes or less. The reaction is permitted to go to completion(approximately 5 minutes).

[0121] Pyridoxal 5 phosphate (PLP) has the ability to modify hemoglobinby introducing a negative charge near a penultimate beta chain histidineresidue and by removing a positive charge at the amino terminal end ofthe same chain. These charge changes stabilize a new molecularconfiguration that is similar to the hemoglobin-DPG (diphosphoglycerate)complex. Significantly, the hemoglobin of this new configuration has anoxygen affinity resembling that of native hemoglobin within the redcell. The product may have one or two PLP molecules attached pertetramer. Although prior PLP-hemoglobin preparations had a satisfactoryoxygen affinity profile, the intravascular retention time was too shortto permit such preparations to be acceptable as a resuscitation fluid.Additionally, they were found to cause osmotic diuresis.

[0122] Accordingly, after the cross-linking reaction has been completed,pyridoxal 5 phosphate (PLP) is added to the dSFH preparation. The PLP isreduced with sodium borohydride and then permitted to react with thecross-linked dSFH and to form FXSFH-pyridoxal-5′-phosphate (FXSFH-PLP)using the methods described by Benesch et al. (Benesch et al.,Biochemistry 11:3576 (1972); Benesch et al., Biochem. Biophys. Res.Commun. 63(4): 1123-9 (1975); Benesch et al., Methods Enzymol. 76:147-59(1981); Benesch et al., J. Biol. Chem. 257(3):13204 (1982); Schnackerzet al., J. Biol. Chem. 258(2):872-5 (1983), all of which references areincorporated by reference in their entirety) with the change that allreagents are free of endotoxin and oxygen and the reaction occurs in theabsence of oxygen.

[0123] Although the properties of deoxygenated stroma-free humanhemoglobin benefit from the above-described pyridoxal 5 phosphatereaction, deoxygenated stroma-free bovine hemoglobin does not requirethis step.

[0124] I. Equilibration

[0125] FXSFH can be equilibrated with lactated Ringers solution. Afterequilibration, the solution is sterile filtered into suitable infusioncontainers. Infusion containers suitable for use in the presentinvention include, but are not limited to, sterile IV bags. Preferredinfusion containers prevent gas exchange (i.e., impermeable to oxygen)and the FXSFH is stored in the absence of oxygen. This is expected toprevent the heme oxidation to form methemoglobin.

[0126] J. Formulations of Blood Substitute Compositions

[0127] The FXSFH of the present invention can be formulated into a bloodsubstitute. Such formulations can include other components in additionto the FXSFH. For example, a parenteral therapeutic composition cancomprise a sterile isotonic saline solution. The formulations can beeither in a form suitable for direct administration, or in aconcentrated form requiring dilution prior to administration. Theformulations of the present invention can thus contain between 0.001%and 90% (w/v) FXSFH. Suitable compositions can also include 0-200 mM ofone or more buffers (for example, acetate, phosphate, citrate,bicarbonate, or Good's buffers). Salts such as sodium chloride,potassium chloride, sodium acetate, calcium chloride, magnesium chloridecan also be included in the compositions of the invention atconcentrations of 0-2 M. In addition, the compositions of the inventioncan include 0-2 M of one or more carbohydrates (for example, reducingcarbohydrates such as glucose, maltose, lactose or non-reducingcarbohydrates such as sucrose, trehalose, raffinose, mannitol,isosucrose or stachyose) and 0-2 M of one or more alcohols or polyalcohols (such as polyethylene glycols, propylene glycols, dextrans, orpolyols). The FXSFH of the present invention can also contain 0.005-1%of one or more surfactants and 0-200 mM of one or more chelating agents(for example, ethylenediamine tetraacetic acid (EDTA), ethyleneglycol-bis (beta-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA),ophenanthroline, diethylamine triamine pentaacetic acid (DTPA also knownas pentaacetic acid) and the like). The compositions of the inventioncan also be at about pH 6.5-9.5.

[0128] The FXSFH of the present invention may contains 0-300 mM of oneor more salts, for example chloride salts, 0-100 mM of one or morenon-reducing sugars, 0-100 mM of one or more buffers, 0.01-0.5% of oneor more surfactants, and 0-150 mM of one or more chelating agents. In astill further embodiment, the composition contains 0-150 mM NaCl, 0-10mM sodium phosphate, and 0.01-0.1% surfactant, and 0-50 μM of one ormore chelating agents, pH 6.6-7.8. The formulation may contain 5 mMsodium phosphate, 150 mM NaCl, 0.025% to 0.08% polysorbate 80, and 25 μMEDTA, pH 6.8-7.6.

[0129] Additional additives to the formulation can includeanti-bacterial agents, oncotic pressure agents (e.g. albumin orpolyethylene glycols) and other formulation acceptable salts, sugars andexcipients known in the art. Each formulation according to the presentinvention can additionally comprise constituents including carriers,diluents, fillers, salts, and other materials well-known in the art, theselection of which depends upon the particular purpose to be achievedand the properties of such additives which can be readily determined byone skilled in the art.

[0130] The compositions of the present invention can be formulated byany method known in the art. Such formulation methods include, forexample, simple mixing, sequential addition, emulsification,diafiltration and the like.

[0131] II. Considerations for Production of FXSFH

[0132] A. Elimination or Reduction of Endotoxin Contamination

[0133] Serum lipases, such as lipase A, do not inactivate endotoxinsbound to the hemoglobin molecule. Therefore, endotoxins remain activetoxins when taken up by the hepatocyte metabolizing the hemoglobin.Friedman, H. I. et al. reported triad hepatoxicity in a rat modelconsistent with this theory (See, Friedman, H. I. et al., Lab Invest39:167-77 (1978).

[0134] Rausch et al. (U.S. Pat. No. 5,084,558) have reported asubstantially endotoxin-free hemoglobin blood substitute. Colpan et al.(U.S. Pat. No. 5,747,663) have reported a process for reducing orremoving endotoxins from a cellular lysate solution. Wainwright et al.(U.S. Pat. No. 5,627,266) have described an endotoxin binding proteinimmobilized to a solid support and the use of this molecule in theremoval of endotoxins from solution.

[0135] Under one preferred embodiment, the elimination of contaminationwith endotoxins is ensured by preventing the addition of endotoxins tothe chemical processes of the present invention. Typically, endotoxinsare added inadvertently by using endotoxin contaminated water.Generally, researchers are more concerned with sterility thanendotoxins. Measurement of endotoxins is difficult, and standard LALbinding assays do not work in the presence of hemoglobin. Indeed,because endotoxin binds strongly to hemoglobin, endotoxin levels cannotbe accurately measured using the LAL assay in the presence ofhemoglobin.

[0136] Water is the most likely candidate for introduction of endotoxinsbecause researchers have long recognized that increased number of stepsin the preparation of hemoglobin increased the level of toxicity.Preparations using dialysis and filtration methods could easily haveexposed the hemoglobin to a thousand volumes of water/buffercontaminated with endotoxin.

[0137] The water and the reagents used in the present invention must besubstantially free from endotoxin contamination. Preferably, the waterand the reagents used in the present invention are completely free fromendotoxin contamination. Preparation of FXSFH in the absence ofendotoxin is extremely difficult to prepare on the bench top, but in aclosed system dedicated to FXSFH manufacture, exclusion of endotoxinwould be easier.

[0138] One way to reduce the risk of endotoxin contamination is toreduce the amount of water and reagent buffers exposed to the hemoglobinpreparation. Therefore, under one preferred embodiment of the presentinvention, the hemoglobin preparations are made using counter-flow orcounter-current dialysis for equilibration of buffers and/or removal ofreaction products. Counter flow dialysis methods are suitable for use inthe present invention are commercially available (e.g., VariPerm M,bitop, Witten (see, e.g., Schwarz, T. et al, Electrophoresis15:1118-1119 (1994)), Spectrum Laboratories, Inc., Laguna Hills, Calif.,etc.). It is estimated that the hollow fiber technique will yield aFXSFH preparation that has a 100 fold reduction in the amount ofendotoxin as compared to standard synthesis techniques.

[0139] B. Reduction or Elimination of Contaminating Phosphate

[0140] Because inorganic phosphate interferes with the cross-linkingreaction, it needs to be removed from the hemoglobin in order for thereaction to provide a satisfactory yield. This can be accomplished usinga suitable exchange resin with chloride ion. A buffer must be providedif there is a substantial change in the exchange of the phosphate ionfor the chloride ion. Preferably, phosphate buffers are not employedduring any of the processing steps of the present invention.

[0141] Under one embodiment of the present invention, the dSFH solutionis substantially free from inorganic phosphate. Preferably, the dSFHsolution of the present invention is free from inorganic phosphate. Oneway of removing inorganic phosphate from the dSFH solution is to passthe SFH solution over an ion exchange matrix equilibrated with chloride.Such a process removes phosphate by competing with phosphate for theaspirin binding site of hemoglobin. This is done in a nitrogenatmosphere. The solution is then concentrated to the desired 10% rangeand cross-linked using the BDBF cross-linker, at standard atmosphericpressure. If human hemoglobin is used, then the reaction with pyridoxal5 phosphate and borohydride is carried out under nitrogen in the absenceof oxygen.

[0142] Preferably, any ion removal or buffer equilibration is performedusing counter flow dialysis so as to prevent accumulation of endotoxinin the subsequent product. The material is then sterile filtered into asuitable container.

[0143] A second problem has been reported to occur in the preparation ofcross-linked hemoglobin for infusion as the phosphate ion must bereplaced prior to infusion to prevent binding a buffered species inplasma.

[0144] Oxygen affinity of the hemoglobin derivative of the presentinvention can be measure using the Hemoxyalayser™ (TCS-Medical Products)or the Gill cell described by Dolman et al., Anal. Biochem. 87:127(1978), incorporated by reference in its entirety.

[0145] C. Nitrous Oxide Regulation of Arterial Blood Supply

[0146] Nitrous oxide is an important regulator of the arterial perfusionof any tissue. Nitrous oxide is synthesized and released by theendothelium in the arterial wall and binds to the hemoglobin in redblood cells. When a tissue is receiving too much oxygen, nitrous oxideis not released and the arterial wall muscle contracts making the vesseldiameter smaller, thus decreasing perfusion. When demand for oxygenincreases, the desaturated hemoglobin releases nitrous oxide, whichcauses vasodilatation. The nitrous oxide control of arterial perfusionworks over small distances in the arterial supply. Because nitrous oxidebinds to hemoglobin inside the red blood cell, it is expected that thenitrous oxide will bind FXSFH as well.

[0147] It has been observed that FXSFH infusion causes vasoconstrictionof the blood vessels, resulting in extremely high blood pressures in theaffected areas. This can make the affected blood vessels very porous,and the FXSFH solution can leak into the surrounding tissues causing thetissues to turn purple. In rabbit models, transfusion of FXSFH throughthe ear vein has caused cerebral vasculature ischemia and death.Therefore, it is important to minimize the impact of administration ofFXSFH on the arterial system during administration.

[0148] Under a preferred embodiment of the present invention, nitrousoxide or a vasoactive agent such as verapamil, Atenocard, etc., isadministered to the patient prior to FXSFH infusion. This is intended toensure that the arterial system is minimally changed during infusion.Nitrous oxide and verapamil are preferred vasoactive agents.

[0149] Under another preferred embodiment, the infusion rate of theFXSFH solution is slowed down to prevent substantial changes in thearterial system of the patient. Slow channel calcium blockers (or aselective inhibitor of cyclic guanosine monophosphate (cGMP)-specificphosphodiesterase type 5 (PDE5), such as sildenafil citrate) may also behelpful in the prevention of the severe vasoconstriction. However, aslower infusion rate may not be preferred with respect to a traumapatient.

[0150] D. Packaging and Storage of FXSFH

[0151] The FXSFH of the present invention may be stored in conventional,and preferably oxygen impermeable containers (for example, stainlesssteel tanks, oxygen impermeable plastic bags, or plastic bagsoverwrapped with low oxygen permeably plastic bags wherein an oxygenscavenger is placed between the internal plastic bag and the overwrappedplastic bag. In another embodiment, the storage stable hemoglobinsolutions can be stored in oxygen permeable or oxygen impermeable(“anoxic”) containers in an oxygen controlled environment. Such oxygencontrolled environments can include, for example, glove boxes, glovebags, incubators and the like. Preferably the oxygen content of theoxygen controlled environment is low relative to atmospheric oxygenconcentrations (see, Kandler, R. L. et al., U.S. Pat. No. 5,352,773;herein incorporated by reference). In a preferred embodiment, the FXSFHof the present invention will be packaged in sealed Tyvek®, or Mylar®(polyethylene terephthalate) bags or pouches. In a second preferredembodiment, the FXSFH of the present invention will be lyophilized andstored as a powder.

[0152] The preparations may be stored at room or elevated temperature(Kandler et al., PCT Publication No. WO 92/02239; Nho, PCT PublicationNo. WO 92/08478, both herein incorporated by reference), or morepreferably under refrigeration.

[0153] In one embodiment, one or more antioxidants such as ascorbate(Wiesehahn, G. P. et al., U.S. Pat. No. 4,727,027; Kerwin, B. D. et al.,U.S. Pat. No. 5,929,031); gluathione, acetylcsyteine, methionine,tocopherol, butyl hydroxy toluene, butyl hydroxy anisole, or pholiccompounds. (Osterber et al., PCT Publication No. WO 94/26286; Kerwin, B.D. et al., U.S. Pat. No. 5,929,031) may be added to further stabilizethe preparation (all such references herein incorporated by reference).

[0154] Alternatively, and more preferably, the FXSFH of the presentinvention will be lyophilized and stored as a powder, or will bepackaged in sealed Tyvek®, or Mylar® (polyethylene terephthalate) bagsor pouches. Packaging such Kerwin, B. D. et al., U.S. Pat. No.5,929,031, herein incorporated by reference).

[0155] In a preferred embodiment, the FXSFH of such storage containerswill be subjected to irradiation or other sterilization treatmentsufficient to extend the shelf-life of the compositions.

[0156] III. Pharmaceutical Uses of the Compositions of the PresentInvention

[0157] The FXSFH of the present invention may be used to formpharmaceutical compositions that may be administered to recipients, forexample, by infusion, by intravenous or intra-arterial injection, or byother means.

[0158] The FXSFH formulations of the present invention can be used incompositions useful as substitutes for red blood cells in anyapplication that red blood cells are used. Such compositions of thepresent invention formulated as red blood cell substitutes can be usedfor the treatment of hemorrhage where blood volume is lost and bothfluid volume and oxygen carrying capacity must be replaced. Moreover,because the FXSFH of the present invention can be made pharmaceuticallyacceptable, the formulations of the present invention can be used notonly as blood substitutes that deliver oxygen but also as simple volumeexpanders that provide oncotic pressure due to the presence of the largehemoglobin protein molecule. The FXSFH of the present invention can thusbe used as replacement for blood that is removed during surgicalprocedures where the patient's blood is removed and saved for reinfusionat the end of surgery or during recovery (e.g., acute normovolemichemodilution or hemoaugmentation, etc.).

[0159] A typical dose of the FXSFH of the present invention as a bloodsubstitute is from 10 mg to 5 grams or more of extracellular hemoglobinper kilogram of patient body weight. Thus, a typical dose for a humanpatient might be from a few grams to over 350 grams. It will beappreciated that the unit content of active ingredients contained in anindividual dose of each dosage form need not in itself constitute aneffective amount since the necessary effective amount could be reachedby administration of a plurality of administrations as injections, etc.The selection of dosage depends upon the dosage form utilized, thecondition being treated, the particular purpose to be achieved accordingto the determination of the ordinarily skilled artisan in the field.

[0160] Administration of the FXSFH of the present invention can occurfor a period of seconds to hours depending on the purpose of thehemoglobin usage. For example, as a blood delivery vehicle, the usualtime course of administration is as rapid as possible. Typical infusionrates for hemoglobin solutions as blood replacements can be from about100 ml to 3000 ml/hour. However, when used to stimulate hematopoiesis,administration can last only seconds to five minutes and thereforeadministration rates can be slower because the dosage of the FXSFH ofthe present invention may be much less than dosages that can be requiredto treat hemorrhage.

[0161] In a further embodiment, the FXSFH of the present invention canbe used to treat anemia, by providing additional oxygen carryingcapacity in a patient that is suffering from anemia, by stimulatinghematopoiesis, and by serving as an adjuvant to erythropoietin therapy.Likewise, the FXSFH of the present invention can be used to provideadditional oxygen carrying capacity to an individual (such as anathelete, soldier, mountaineer, aviator, smoke victim, etc.) desiringsuch additional oxygen carrying capacity. The formulations of thepresent invention thus are useful in treating hypoxia and ischemia.

[0162] In addition, because the distribution in the vasculature of theFXSFH of the present invention is not limited by viscosity or by thesize of red blood cells, the compositions of the present invention canbe used to deliver oxygen to areas that red blood cells cannotpenetrate. These areas can include any tissue areas that are locateddownstream of obstructions to red blood cell flow, such as areasdownstream of thrombi, sickle cell occlusions, arterial occlusions,angioplasty balloons, surgical instrumentation and the like.

[0163] In a further embodiment, the FXSFH of the present invention canbe used to treat excess nitric oxide concentrations. Excess nitric oxidehas been implicated in conditions ranging from hypotension to septicshock. Because the hemoglobin of the present invention can bind nitricoxide and other non-oxygen ligands as well as oxygen, the FXSFH of thepresent invention can be used to effect the removal excess nitric oxide(or such non-oxygen ligands), or to attenuate the concentration of suchnitric oxide and non-oxygen ligands. Such treatment can be accomplishedeither by administration of FXSFH to the patient, or in an ex vivomanner (as by contacting the patient's blood with immobilized FXSFH,etc.).

[0164] The FXSFH of the present invention contains iron, and as such,may be detected via MRI (magnetic resonance imaging). Thus, in a furtherembodiment, the present invention contemplates the use of FXSFH as animaging agent.

[0165] Although humans have four main red cell antigens (A, B, O andRh), accounting for 12 main blood types, non-human animals exhibit fargreater blood type diversity. The existence of larger numbers of bloodtypes has complicated the use of donated blood in non-human animaltransfusions (Hale, A. S., Vet Clin North Am Small Anim Pract25:1323-1332 (1995); Harrell, K. A., et al., Vet Clin North Am SmallAnim Pract 25:1333-1364 (1995), both references herein incorporated byreference. The FXSFH formulations of the present invention, which can beused regardless of the blood type of the recipient, thus findsadditional utility as a blood substitute for non-human animals (e.g.,dogs, horses, cats, etc.).

[0166] The present invention also concerns implantable delivery devices(such as cartridges, implants, etc.) that contain FXSFH, and that arecapable of releasing FXSFH into the circulation in response to a sensedneed for increased oxygen carrying capacity. In one embodiment, suchdevices will deliver FXSFH at a constant rate, so as to facilitateerythropoiesis (either alone, or in combination with erythropoietin). Ina second embodiment, the devices will be controlled by sensing means(such as electronic probes of hemoglobin, O₂ level, CO₂ level, etc.) soas to deliver FXSFH at a rate commensurate with the patient's oxygencarrying capacity needs. Such sensing means may be themselvesimplantable, or part of the implanted device, or may be locatedextracorporeally. In a further sub-embodiment, such devices may be usedto accomplish or facilitate the hemo-diagnosis of individuals.

[0167] IV. Non-Pharmaceutical Uses of the Compositions of the PresentInvention

[0168] The FXSFH of the present invention may also be used to formnon-pharmaceutical compositions that can be used, for example, asreference standards for analytical instrumentation needing suchreference standards, reagent solutions, control of gas content of cellcultures; for example by in vitro delivery of oxygen to a cell culture,and removal of oxygen from solutions.

[0169] Additionally, the FXSFH of the present invention may be used tooxygenate donated tissues and organs during transport.

[0170] In a preferred non-pharmaceutical use, the FXSFH of the presentinvention may be used to scavenge endotoxin from surfaces or liquids.The invention thus contemplates devices, such as cartridges, filters,beads, columns, tubing, and the like that contain the FXSFH of thepresent invention. Liquids, such as water, saline, culture medium,albumin solutions, etc., may be treated by passage over or through suchdevices in order to remove endotoxin that may be present in suchliquids, or to lessen the concentration of endotoxin present in suchliquids. The FXSFH of such devices is preferably immobilized (as byaffinity, ionic, or covalent bonding, etc.) to solid supports present insuch devices. In one sub-embodiment, the FXSFH is bound to beads thatmay be added to the liquids being treated, and then subsequently removed(as by filtration, or affinity immobilization). In a furthersub-embodiment, the beads may be of ferromagnetic or paramagnetic metal,or may be themselves magnetic, such that they may be readily separatedfrom the treated liquid by magnetic means.

[0171] Likewise, the FXSFH of the present invention may be adsorbed orbound to toweling, air filters, etc. so that endotoxin present onsurfaces or in air may be removed or its concentration lessened.

[0172] In a similar manner, the FXSFH of the present invention can beused to remove oxygen from solutions requiring the removal of oxygen,and as reference standards for analytical assays and instrumentation.

[0173] The FXSFH of the present invention can also be used in vitro toenhance cell growth in cell culture by maintaining oxygen levels.

[0174] It will be apparent to those skilled in the art that variousmodifications may be made in the present invention without departingfrom the spirit and scope of the present invention. It will beadditionally apparent to those skilled in the art that the basicconstruction of the present invention is intended to cover anyvariations, uses or adaptations of the invention following, in general,the principle of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains. Therefore, it will be appreciatedthat the scope of this invention is to be defined by the claims appendedhereto, rather than the specific embodiments which have been presentedas examples.

1-18. (canceled).
 19. A preparation of non-pyrogenic, endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin produced by the processcomprising the steps of: (A) removing endotoxin from a preparationcontaining red blood cells; (B) removing oxygen from said preparationcontaining red blood cells, wherein said oxygen is removed bycentrifuging the red blood cells under a vacuum sufficient to removeoxygen from the preparation; (C) lysing the red blood cells. 20.(canceled)
 21. The preparation of non-pyrogenic, endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin of claim 19, whereinsaid process additionally comprises the steps of: (D) separatinghemoglobin from the stroma of said lysed red blood cells; and (E)cross-linking said separated hemoglobin.
 22. The preparation ofnon-pyrogenic, endotoxin-free, stroma-free, cross-linked tetramerichemoglobin of claim 21, wherein said process step (A) additionallycomprises washing surfaces and equipment that will come into contractwith the cross-linked hemoglobin with a dilute solution of a hemoglobin.23. The preparation of endotoxin-free, stroma-free, cross-linkedtetrameric hemoglobin of claim 19, wherein said process step (B)comprises centrifuging a solution of said cells under vacuum at a speedsufficient to produce a force greater than the surface tension of thesolution.
 24. The preparation of endotoxin-free, stroma-free,cross-linked tetrameric hemoglobin of claim 21, wherein said red bloodcells are human red blood cells.
 25. The preparation of endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin of claim 21, whereinsaid red blood cells are bovine or porcine red blood cells.
 26. Thepreparation of endotoxin-free, stroma-free, cross-linked tetramerichemoglobin of claim 21, wherein said preparation additionally contains apharmaceutically acceptable carrier.
 27. A preparation of non-pyrogenic,endotoxin-free, stroma-free, cross-linked tetrameric hemoglobin producedby the process comprising the steps of: (A) removing endotoxin from apreparation containing red blood cells; (B) lysing red blood cells; and(C) removing oxygen from hemoglobin of said lysed red blood cells,wherein said oxygen is removed by centrifuging the red blood cells undera vacuum sufficient to remove oxygen from the preparation. 28.(canceled).
 29. The preparation of non-pyrogenic, endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin of claim 27, whereinsaid process additionally comprises the steps of: (D) separatinghemoglobin from the stroma of said lysed red blood cells; and (E)cross-linking said separated hemoglobin.
 30. The preparation ofnon-pyrogenic, endotoxin-free, stroma-free, cross-linked tetramerichemoglobin of claim 27, wherein said process step (A) additionallycomprises washing surfaces and equipment that will come into contactwith the cross-linked hemoglobin with a dilute solution of a hemoglobin.31. The preparation of endotoxin-free, stroma-free, cross-linkedtetrameric hemoglobin of claim 27, wherein said process step (C)comprises centrifuging a solution of said cells under vacuum at a speedsufficient to produce a force greater than the surface tension of thesolution.
 32. The preparation of endotoxin-free, stroma-free,cross-linked tetrameric hemoglobin of claim 27, wherein said red bloodcells are human red blood cells.
 33. The preparation of endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin of claim 27, whereinsaid red blood cells are bovine or porcine red blood cells.
 34. Thepreparation of endotoxin-free, stroma-free, cross-linked tetramerichemoglobin of claim 27, wherein said preparation additionally contains apharmaceutically acceptable carrier. 35-48. (canceled).
 49. A method ofincreasing the oxygen carrying capacity of an individual which comprisesadministering to said individual a non-pyrogenic, endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin administered bytransfusion or injection, wherein said non-pyrogenic, endotoxin-free,stroma-free, cross-linked tetrameric hemoglobin is produced by a processcomprising the steps: (A) removing endotoxin from a preparationcontaining red blood cells; (B) removing oxygen from said preparationcontaining red blood cells, wherein said oxygen is removed bycentrifuging the red blood cells under a vacuum sufficient to removeoxygen from the preparation; and (C) lysing red blood cells. 50-52.(canceled).
 53. The method of claim 49, wherein said processadditionally comprises the steps of: (D) separating hemoglobin from thestroma of said lysed red blood cells; and (E) cross-linking saidseparated hemoglobin.
 54. The method of claim 49, wherein said processstep (A) additionally comprises washing surfaces and equipment that willcome into contact with the cross-linked tetrameric hemoglobin with adilute solution of hemoglobin.
 55. The method of claim 49, wherein saidprocess step (B) additionally comprises centrifuging a solution of saidcells under vacuum at a speed sufficient to produce a force greater thanthe surface tension of the solution.
 56. The method of claim 53, whereinsaid hemoglobin is human hemoglobin.
 57. The method of claim 53, whereinsaid hemoglobin is bovine or porcine hemoglobin. 58-60. (canceled). 61.The method of claim 56, wherein said process step (A) comprises washingsurfaces and equipment that will come into contact with the cross-linkedtetrameric hemoglobin with a dilute solution of hemoglobin.
 62. Themethod of claim 56, wherein said process step (C) additionally comprisescentrifuging a solution of said cells under vacuum at a speed sufficientto produce a force greater than the surface tension of the solution. 63.The method of claim 53, wherein said hemoglobin is human hemoglobin. 64.The method of claim 53, wherein said hemoglobin is bovine or porcinehemoglobin. 65-71. (canceled).